There are two general categories of histochemical specimens. The most common is fixed, paraffin-embedded tissue specimens. These specimens are fixed, (usually using a formalin-based fixative), dehydrated to xylene, embedded in paraffin, sectioned onto a slide, deparaffinized, rehydrated, and stained. The second category includes preparations which are fresh tissues and/or cells, which generally are not fixed with aldehyde-based fixatives. Such specimens are either placed directly on a slide or coverslip, or frozen and sectioned onto slides. Such specimens are then fixed, usually with an alcohol- or acetone-based fixative, and stained. These specimens commonly include biopsy materials which may be analyzed while the surgical procedure is in progress (frozen sections), cytological preparations (including touch preparations and blood smears), and tissues which are to be immunohistochemically analyzed.
Although buffered formaldehyde-based fixative solutions at pH's of 7.0 or greater (e.g., 10% neutral buffered formalin (NBF), 1-4% paraformaldehyde, Zamboni's, Bouin's, B5, Hollande's) provide excellent results for conventional staining methods, these fixatives are problematic for use with antibody staining methods as they covalently modify and cross link the antigens. This often results in the direct alteration of the epitope(s) or epitopic accessibility, either of which prevents antibody recognition and/or binding. Thus, antibodies are unable to detect many antigens present in tissues fixed using aldehyde-based fixatives.
Fresh biopsy specimens, cytological preparations, (including touch preparations and blood smears), frozen sections and tissues for immunohistochemical analysis are commonly fixed in organic solvents (ethanol, methanol, and/or acetone). Although such fixatives do not create problems with antibody recognition due to cross linking, the fixatives do create problems of membrane extraction, nuclear lysis, and loss of antigenicity in specimens. The alcohol fixatives, while improving morphology over acetone-fixed preparations, allow significant extraction and result in greater loss of antigenicity than acetone-fixed tissues. Acetone preserves antigenicity well, but poorly preserves morphology, and allows antigens to diffuse and be extracted. Aldehyde-based fixatives can be used on nonparaffin-embedded specimens to preserve morphology and prevent extraction. However, as stated above, the aldehyde fixatives significantly destroy antigenicity and increase the difficulty of antibody penetration.
A non-aldehyde-based fixative which does not have the disadvantages of the alcohol or acetone fixatives is desirable.